Phage Cocktails Constrain the Growth of Enterococcus

ABSTRACT Phages that infect pathogenic bacteria present a valuable resource for treating antibiotic-resistant infections. We isolated and developed a collection of 19 Enterococcus phages, including myoviruses, siphoviruses, and a podovirus, that can infect both Enterococcus faecalis and Enterococcus faecium. Several of the Myoviridae phages that we found in southern California wastewater were from the Brockvirinae subfamily (formerly Spounavirinae) and had a broad host range across both E. faecium and E. faecalis. By searching the NCBI Sequence Read Archive, we showed that these phages are prevalent globally in human and animal microbiomes. Enterococcus is a regular member of healthy human gut microbial communities; however, it is also an opportunistic pathogen responsible for an increasing number of antibiotic-resistant infections. We tested the ability of each phage to clear Enterococcus host cultures and delay the emergence of phage-resistant Enterococcus. We found that some phages were ineffective at clearing Enterococcus cultures individually but were effective when combined into cocktails. Quantitative PCR was used to track phage abundance in cocultures and revealed dynamics ranging from one dominant phage to an even distribution of phage growth. Genomic characterization showed that mutations in Enterococcus exopolysaccharide synthesis genes were consistently found in the presence of phage infection. This work will help to inform cocktail design for Enterococcus, which is an important target for phage therapy applications. IMPORTANCE Due to the rise in antibiotic resistance, Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy offers an alternative to antibiotics and has shown promise in both in vitro and early clinical studies. Here, we established a collection of 19 Enterococcus phages and tested whether combining phages into cocktails could delay growth and the emergence of resistant mutants in comparison with individual phages. We showed that cocktails of two or three phages often prevented the growth of phage-resistant mutants, and we identified which phages were replicating the most in each cocktail. When resistant mutants emerged to single phages, they showed consistent accumulation of mutations in exopolysaccharide synthesis genes. These data serve to demonstrate that a cocktail approach can inform efforts to improve efficacy against Enterococcus isolates and reduce the emergence of resistance.


Response to reviewer comments for "Phage Cocktails constrain the growth of
Enterococcus" by Stephen Wandro, Pooja Ghatbale, and coauthors (# mSystems00019-22R1) : We thank the reviewers for the time spent reviewing our manuscript and for their helpful suggestions. Their comments have made the manuscript much better during both revisions. We've made several changes to the manuscript text and figures. See below for a point-by-point response to the reviewer's comments (in purple font) and a detailed guide to the manuscript changes. 1. Table 2 is fairly uninformative as is, and is in need of major improvement. A SNP has little meaning by itself. The SNP and position in the gene should be listed (eg., A225G, or G112-, etc.). Figure S3 is not sufficient to provide this information. Instead of SNP, use missense, nonsense, silent, DEL, or INS. If a missense mutation, then the amino acid change and residue number should be listed (eg., Ala48Gln). The table length will likely expand, since some genes may have multiple different mutations. However, this is very important data that is central to the paper.

Response:
We agree, and have substantially modified Table 2 with the suggested additions, including a column focused on the Epa locus as described in Reviewer 1 Response 1 above.

2.
At least a few of the phage resistant mutants in Table 2 should be validated by plaque assay using each of the phages. The story is incomplete without this data.
Response: Thank you for this suggestion. Although we agree that this line of experiments would be informative, colony purified isolates would be required for effective plaque assays, and obtaining these isolates is beyond the scope of our manuscript. As addressed in point 11 below, all sequencing experiments were performed on communities, not bacterial isolates.

Response to Reviewer #2 (Minor Comments):
1. Line 90: Change "would" to "may". If the phages in the cocktail use the same receptor, then a single mutation can give resistance to all the phages.
Response: Thank you for the suggestion. To address this point we changed "would" to "would likely" in Line 90.

2.
Line 92: Change "making" to "for example making". It is more common that phage resistance mutations reduce virulence or some other facet of fitness than resistance to other phages. In fact, you make this point in the Discussion, Line 282.
Response: "for example by" was added to line 92 as suggested.
Response: Specified "both antibiotic and phage resistant" in line 105.

5.
Lines 117-124: You don't say if the 20 phages are novel or already known. If already published or their genome is in Genbank, you need to reference accordingly.  Table S3, together with Genome size and Genbank accession numbers. " Table S3 figure legend now reads: The following phages were isolated and included in these experiments, with the phage name, Family, Genus, Genome size and Genbank accession numbers for the sequencing data we deposited, with the exception of EfV12-phi1, which was already available and which we used in our initial coevolution study Wandro et al 2019 31 . The naming convention for each phage begins with vB_OCPT for virus of bacteria and Orange County Phage Team (OCPT). And the Data Availability section also refers to the genbank accession numbers in Table  S3.

6.
Line 125: Delete "Core". It is must be "Genome alignments.." since you wouldn't know what is a core gene prior to aligning the genomes.
Response: "Core" was removed as suggested.

Response:
Changed "how phages" to "how the phages we isolated". Fig 2: Some numbers on the trees overlap the tree, making it hard to decipher. Move them off to one side. If necessary, cover them up and retype the number in a better location. Also, some font sizes are very tiny. Make larger.

8.
Response: Thank you for your suggestions. We've modified figure 2 to improve readability.

10.
Line 312: Are you saying the patient's names were de-identified? Presumably not, since that would be a HIPAA violation? Perhaps you mean the clinical isolates of Enterococcus were deidentified.
Response: By de-identified, we mean that all personal identifying information from the patients has been removed. We never had access to patient names as part of our study. Definition of de-identified from https://www.edglossary.org/de-identified-data/ : "In education, de-identified data generally refers to data from which all personally identifiable information has been removed." This is now around Line 312, which now reads: "E. faecalis and E. faecium strains were added to our strain collection from the patient population at UC San Diego Health; all personally identifying information was removed." 11. Line 453: It appears you prepped and sequenced gDNA from the culture populations, instead of from individual colony-purified and confirmed phage-resistant strains. Is this true? If so, it should be stated that the cultures may contain multiple strains for clarification.
Response: Your observation is accurate; all sequencing experiments were performed on populations, not colony-purified strains. We have added the following statement starting on Line 468 of the manuscript to clarify this important point: "All sequencing experiments were performed using cultured populations of bacteria as opposed to individual colony-purified strains, therefore each culture likely contains DNA from multiple strain variations." 1st Revision -Editorial Decision Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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